Primary publication: Sherrill et al, 2014 https://doi.org/10.1038/gene.2014.27
Experimental Design:
RNA sequencing was performed to quantify transcripts present in esophageal biopsy specimens obtained from 6 healthy controls (Control) and 10 patients with active EoE (EoE).
Primary publication: Rochman et al, 2014 https://doi.org/10.1038/mi.2014.109
TE-7 cells (a human esophageal epithelial cell line) were stimulated with IL-13 (100 ng/ml) for 2h, 6h, or 24h. Transcript levels were quantified by RNA sequencing. Untreated cells (Control_2h, Control_4h, Control_24h); IL-13-treated cells (IL-13_2h, IL-13_6h, IL-13_24h).
Primary publication: Azouz et al, 2018 https://doi.org/10.1126/scitranslmed.aap9736
A telomerase-immortalized human esophageal epithelial cell line (EPC2 cells) was transfected with either non-silencing control shRNA or SPINK7 shRNA. Cells were grown either in monolayer culture (undifferentiated) or in air liquid interface (ALI) cultures (differentiated). Transcript levels of the following types of cells were determined: cells transfected with non-silencing control shRNA and grown in monolayer culture (Undif_NSC); cells transfected with non-silencing control shRNA grown at the ALI (NSC); cells transfected with SPINK7 shRNA grown at the ALI (SPINK7_silenced).
Primary publication: Sherrill et al, 2014 https://doi.org/10.1038/mi.2013.90
A telomerase-immortalized human esophageal epithelial cell line (EPC2 cells) was grown submerged in monolayer culture (undifferentiated) for 8 days, or were grown submerged in monolayer culture for 8 days followed by 6 days of culture at the air liquid interface (differentiated). In some cases, cells were treated with IL-13 (100 ng/ml) during the 6 days of ALI culture. Transcript levels of the following conditions were assessed by RNA sequencing: Day_0: cells grown submerged for 8 days; Day_6: cells grown submerged for 8 days and then grown at the ALI for 6 additional days; Day_6_IL-13: cells grown submerged for 8 days and then grown at the ALI for 6 additional days in the presence of IL-13.
Primary publication: Blanchard et al, 2006 https://doi.org/10.1172/JCI26679 Blanchard et al, 2007 https://doi.org/10.1016/j.jaci.2007.10.024 Caldwell et al, 2010 https://doi.org/10.1016/j.jaci.2010.01.038
RNA isolated from distal esophageal tissue was subjected to genome-wide transcript profile microarray analysis. Experimental groups: NL: control; CE: chronic esophagitis; EoE: active EoE; EoE+FP+R: fluticasone responder; EoE+FP+NR: fluticasone non-responder.
Primary publication: Caldwell et al, 2014 https://doi.org/10.1016/j.jaci.2014.07.026
RNA isolated from gastric antrum tissue was subjected to genome-wide transcript analysis by microarray analysis. Experimental groups: control and active eosinophilic gastritis (EG).
Primary publication: Caldwell et al, 2014 https://doi.org/10.1016/j.jaci.2007.10.024
Human primary esophageal epithelial cells from patients with EoE were cultured in the presence or absence of IL-13 (100 ng/ml) for 48 h, and the mRNA was subjected to genome-wide transcript profile microarray analysis. The data from the IL-13-stimulated cells were normalized pairwise to unstimulated controls.
Primary publication: Shoda et al, 2018 https://doi.org/10.1016/S2468-1253(18)30096-7
RNA was isolated from distal esophageal tissue of 86 individuals with active EoE. The RNA was subjected to EDP analysis, which quantifies 96 individual gene transcripts. Graphs depict the -deltaCt value -(ct of GAPDH – ct of gene of interest) for each individual. Experimental groups: EoEe1: EoE endotype 1; EoEe2: EoE endotype 2; EoEe3: EoE endotype 3.
Primary publication: Wen et al, 2019 https://doi.org/10.1172/JCI125917
Single-cell transcriptome profililng of esophagus biopsies from patients with active eosinophilic esophagitis (EoE, n=11), in remission (n=6), and without disease (n=5).
Primary publication: Ben Baruch-Morgenstern et al, 2016 https://doi.org/10.4049/jimmunol.1501873
17 samples were analyzed in this experiment. The experiment was designed with 3 replicates for each treatment/genotype/tissue (Dox and no Dox/wildtype and knockout/bone marrow and esophagus), with the exception of the sample wildtype bone marrow no Dox where 1 replicate was dropped due to low hybridization signal. The no Dox and wildtype samples are controls for the treatment and background of the mice. Experimental groups: bm_WT_noDox: bone marrow, wildtype, no Dox; bm_KO_noDox: bone marrow, PirB knockout, no Dox; bm_WT_Dox: bone marrow, wildtype, Dox; bm_KO_Dox: bone marrow, PirB knockout, Dox; eso_WT_Dox: esophagus, wildtype, Dox; eso_KO_Dox: esophagus, PirB knockout, Dox.
RNA sequencing was performed to quantify transcripts present in gastric biopsy specimens obtained from 12 healthy controls (Non-EG) and 9 patients with EG (EG).
RNA sequencing was performed to quantify transcripts present in LDBM Mouse Eosinophils untreated, treated with IL-4, or treated with IL-33 at 1 hour and 4 hours.
Primary publication: Rochman et al, 2020 https://doi.org/10.1016/j.jaci.2020.09.039
For the monolayer cultures, EPC2 cells were seeded at a density of 2.5x10^5 cells/well in KSFM in a 24-well plate. The next day, the medium was replenished. After 24 hours, stimulants were added in a total of 350 to 500mL of fresh medium for 24 hours. IL-13 was added to a final concentration of 100 ng/mL unless otherwise indicated. Omeprazole and esomeprazole were dissolved in DMSO to the stock concentration of 100 mM, aliquoted into 10-mL amounts, and stored at –80°C. PPIs were thawed once and used at a working concentration of 100mM; cells were pretreated with the PPIs for 1 hour before adding IL-13 to the medium. GNF-351 was dissolved in DMSO to a 20 mM stock concentration. The stock was aliquoted in 10-mL doses and stored at –80°C. Cells were pretreated with GNF-351 at a final concentration of 2mM for 30 minutes before adding PPIs.
RNA sequencing was performed with high-quality RNA (RNA integrity number > 8) by using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria, catalog no 015.96).
Libraries were diluted to final concentrations of 5 nM and sequenced on a HiSeq 4000 Illumina sequencing machine at the Genomics and Cell Characterization Core Facility at the University of Oregon with 100- to 150-bp-length single reads.
Multiplex analysis was performed by Eve Technologies (Calgary, Canada) using the Human Cytokine Array/Chemokine Array 65-Plex Panel (HD65).
Primary publication: Zuo et al. 2010, JI https://www.jimmunol.org/content/185/1/660
Bi-transgenic mice (CC10-iIL-13) were generated in which IL-13 was expressed in a lung-specific manner that allowed for external regulation of the transgene expression, as previously described(13). RNA from the esophagus or lung of IL-13 inducible transgenic mice obtained after 30 days of DOX or NO-DOX treatment was subjected to gene chip analysis using MOE 430 2.0 chips as previously reported (15). Gene transcript levels were determined using algorithms in the Microarray Analysis Suite and GeneSpring software (Silicon Genetics). For comparison with human EE, genes were translated to human homologues represented on the human U133 chip using GeneSpring software and compared to microarray results previously described(8, 15). Welch T test and fold change cut-off were performed. The correlation between IL-13 expression and numbers of dysregulated genes were also analyzed using GeneSpring software. 2 NO-DOX samples and 3 DOX samples MOE 430 2.0 chips - Affymetrix Composition of probe sets can be identified/analyzed on NetAffx https://www.affymetrix.com/site/login/login.affx?toURL=/analysis/netaffx/index.affx
References: 8 - https://pubmed.ncbi.nlm.nih.gov/18073124/ 13 - https://pubmed.ncbi.nlm.nih.gov/14757645/ 15 - https://pubmed.ncbi.nlm.nih.gov/16453027/